The primary form of PTH, which is stored and secreted, contains 84 amino acids.
PTH is initially synthesized as a precursor, preProPTH.
Two proteolytic cleavages produce the ProPTH and the secreted form of PTH.
The proPTH sequence contains six extra amino acids at the N-terminus.
Conversion of ProPTH to PTH occurrs about 15 to 20 min after biosynthesis at about the time ProPTH reached the Golgi apparatus.
The Structure of the Pre-Peptide
Evidence that the translational product of PTH mRNA was larger than ProPTH was initially obtained by translation of a crude preparation of bovine parathyroid RNA in the wheat germ cell-free system.
The primary translational product migrated slower than ProPTH when analyzed by electrophoresis on either acidic-urea or sodium dodecyl sulfate-containing acrylamide gels.
At that time, a similar phenomenon had been observed only for myeloma light chains.
In further studies, preProPTH was shown to be synthesized in cell-free systems of reticulocyte lysates.
Translation of human parathyroid RNA also produced an analogous preProPTH.
The observation that the carboxyl terminal peptides of bovine PTH and preProPTH were identical indicated that the extra amino acids in preProPTH were at the amino terminus.
This was confirmed by incorporating selected radioactive amino acids into preProPTH and determining the location of the radioactivity by automated Edman degradation.
By analyzing overlap of these radioactive amino acids with those in ProPTH, the length of the bovine pre-peptide was shown to be 25 amino acids.
The entire sequence of the bovine pre-peptide was determined eventually by this microsequencing technique and was later confirmed by structural studies of both the bovine PTH cDNA and gene.
The sequence of human pre-peptide was also partially determined by this microsequencing technique.
The complete amino acid sequence was derived from the human PTH cDNA sequence and later confirmed by the determination of the structure of the human gene.
The amino acid sequence of the rat pre-peptide was derived from the sequence of the rat PTH gene and partially by analysis of cloned rat PTH cDNA.
The amino acid sequences of the pre-peptides show that the human and bovine pre-peptides are 80% homologous while the rat sequence is 64% homologous to the bovine and human.
This is somewhat lower than the homology of 89 and 77% in the Pro and PTH regions for bovine/human and rat/bovine-human, respectively.
The fact that the pre-peptide is less conserved than the rest of the molecule is consistent with pre-peptides or signal peptides of many eukaryotic proteins.
General structural features of the signal peptides are a central hydrophobic core and, in many cases, charged amino acids at the N-terminal and C-terminal ends of the central core.
These features are largely retained in the pre-peptides of the three preProPTH molecules.
Only conservative changes are present within the central core of uncharged amino acids from amino acids 10 to 21.
Conversion of PrePro to ProPTH
The removal of the pre-peptide to produce ProPTH is mediated by an enzyme associated with microsomes.
In reticulocyte and wheat germ systems that contain little or no microsomal membranes, the primary transcriptional product of PTH mRNA is preProPTH.
Addition of microsomal membranes from dog pancreas or chicken oviduct results in the synthesis of ProPTH.
The first evidence that pre or signal peptides function by binding to a limited number sites in the microsomal membrane was obtained by studies on a synthetic prePro-peptide of bovine preProPTH.
The identification of the signal recognition particle as a signal peptide receptor, later on, confirmed this mechanism for most secreted and membrane proteins.
The pre peptide of preProPTH is rapidly degraded after its proteolytic cleavage from preProPTH.
In studies of PTH biosynthesis in intact cells, no labeled pre-peptide could be detected.
The proteolytic removal of the pre-peptide probably occurs before completion of the ProPTH nascent chain, since preProPTH is difficult to detect in intact cells.
Homology of the Mature PTH
The mature PTH has been determined or predicted by the cDNAs in several species.
A comparison of the amino acid sequences of PTH from several species revealed high conservation of the protein amongst all species apart from gallus.
In addition, three relatively conserved regions could be observed.
The first two regions comprise the biologically active region of PTH and would be expected to be conserved.
The addition or loss of a single amino acid at the amino terminus greatly reduces biological activity, and the region is involved in binding of PTH to the receptor.
In addition there is a region of conservation at the C-terminal region that is itself of interest, particularly since this region may have a separate biological effect at least on osteoclasts.
Analyses of the silent changes that occur between the nucleotide sequences suggest that the conservation in the C-terminal region may be related to pre-translational events.
Analysis by Perler et al described replacement changes that result in changes in amino acids and silent changes that do not alter the encoded amino acid.
The PTH mRNA
Bovine preProPTH mRNA was initially more extensively characterized than the mRNAs from the other species.
Preparations of bovine parathyroid RNA were obtained that contained about 50% PTH mRNA as estimated by gel electrophoresis and RNA excess hybridization to radioactive cDNA.
The size of the mRNA was estimated to be about 750 nucleotides by sucrose gradient centrifugation.
About two thirds of the translatably active mRNA was retained by oligo(dT) cellulose, and the sizes of the poly(A) extension was broadly distributed around an average size of 60 adenylate residues, though this may be an under estimation of the actual size.
While not directly determined, PTH mRNA probably contains a 7-methylguanosine cap since the translation of PTH mRNA was inhibited by 7-methylguanosine-5'-phosphate.
The human and bovine PTH mRNAs appear to be heterogeneous at the 5’ terminus (see section on genes).
The sizes of the rat and human PTH mRNAs have been determined by Northern blot analysis to be about 800 and 850 nucleotides, respectively.
Therefore, PTH mRNAs are typical eukaryotic mRNAs that contain a 7-methyguanosine cap at the 5’ terminus and a polyadenylic acid (poly A) stretch at the 3’ terminus.
The PTH mRNAs are twice as long as necessary to code for the primary translational product, due to 5' and 3' untranslated regions at both ends of the mRNA.
Cloning of the PTH cDNAs
To date the sequence of the full cDNA of rat, man, dog, cat (un published), cow, pig, and chicken and the partial sequence of horse and non human primates have been determined.
The cDNA of mouse PTH was determined from the genomic PTH sequence. In addition, the hypothalamus PTH cDNA was sequenced after the PTH mRNA had been detected in neuronal tissue.
The first PTH cDNAs identified were the DNAs complementary to bovine and human PTH mRNA that had been cloned into the Pst 1 site of pBR322 by the homopolymer extension technique.
The rat PTH cDNA was cloned by the Okayama and Berg method. The bovine mRNA was isolated from normal parathyroid glands, and the human mRNA was isolated from parathyroid adenomas.
The sequence of the rat mRNA has been derived partially from the rat cDNA and from the sequence of the cloned gene.
Kronenberg et al initially determined the sequence of a bovine cDNA clone, pPTHml, which contained about 60% of the PTH mRNA, including the entire region coding for pre-ProPTH. Restriction analysis of near full-length double-stranded cDNA, synthesized enzymatically from partially purified bovine PTH mRNA, indicated that about 200 nucleotides from the 3’ untranslated region were missing in the clone.
Analysis of several additional bovine PTH cDNA clones and the sequencing of cDNA of the 5’ terminus of PTH mRNA, which was synthesized by extension of a primer with reverse transcriptase, provided the full bovine DNA sequence.
Nucleotide sequences of the parathyroid (PTH) gene of 12 species of non-human primates belonging to suborder Anthropoidea were characterized.
The deduced amino acid sequences of exons II and III of the PTH gene of the 12 species of non- human primates was compared to the human PTH and revealed no amino acid substitution in the mature PTH among orangutans, chimpanzees, and humans.
The results indicated that the PTH gene is highly conserved among primates, especially between great apes and humans.
The 5’ end of the bovine mRNA sequence, which was determined by sequencing DNA complementary to the 5’ end of PTH mRNA produced by primed reverse transcription, produced multiple 5’ termini of the mRNA. The heterogeneity at the beginning of the 5’ end of the mRNA was confirmed by S1 nuclease mapping.
The longest reverse transcribed cDNA was isolated and sequenced.
Surprisingly, this cDNA contained a canonical TATA sequence at the beginning, which was in the proper position to direct the transcription of the shorter mRNAs.
This result suggested that a second TATA sequence would be present 5’ to the one detected in the cDNA and would direct the synthesis of the longer mRNAs.
The predicted second TATA sequence was discovered when the gene was sequenced.
The 5’ end of the rat PTH mRNA was also analyzed by S1 nuclease mapping and was less heterogeneous than the bovine mRNA.
The single species of rat PTH mRNA corresponded to the larger of the bovine mRNAs.
The size of the human mRNA, based on the cDNA sequence, is about 100 nucleotides longer than the bovine and rat mRNAs. Northern blot analysis of the mRNAs was consistent with these predicted sizes.
The 3' untranslated region (UTR) of the avian PTH mRNA is 1236 nt long, much larger than any of the PTH mRNA 3'-UTRs.
In general the difference in size in the PTH mRNA of the different species primarily results from the difference in the size of the 3’-UTR.
The significance of this finding has not been studied.
The overall nucleotide compositions of the cDNAs are similar.
All the sequences are A-T rich. The 3’ noncoding region has a particularly large portion of A and T, ranging from 68 to 74%, making it an AU rich element (ARE).
The rat sequence differs from the other sequences in that the 5’ noncoding region is only 50% A and T compared to 63 to 65% for the human and bovine.
Homology of the cDNA Sequences
Gaps have been introduced in the 5’ and 3’ untranslated regions to maximize homology.
This PTH mRNA is significantly longer than the other cloned cDNAs and is the least preserved compared to the other species, even in the coding sequence.
Comparison of the sequences show that human and macaca; canis and felis; rat and mouse; and bovine and pig are the most similar to each other.
The lowest homology is seen when the sequence of gallus PTH mRNA is compared to each of the other sequences, even in the translated coding region of the mRNA that is, as expected, the most conserved region. The coding sequences of the other species are the most preserved as expected.
The 5’-UTR is relatively well conserved with homologies about 15% less than the coding region.
The 3’-UTR is the least conserved region.
Interestingly, a 26 nt cis acting functional protein binding element at the distal region of the 3' UTR is highly conserved in the PTH mRNA 3'-UTRs of rat, mouse, man, dog and cat.
In the 26 nt element, the identity amongst species varies between 73 and 89%.
In particular, there is a stretch of 14 nt within the element that is present in all five species.
We have previously characterized this distal protein binding element in the rat PTH mRNA 3’-UTR as a cis-acting sequence that determines the stability of the PTH mRNA and its regulation by calcium and phosphate (P). In addition, a 22 nt protein binding element in the 3' UTR was also identified in bovine and porcine, as well as human, non-human primates, equus, canis and felis, but not in rat and mouse.
The functionality of the proximal element remains to be determined.
The conserved sequences within the 3'-UTR suggest that the binding elements represent a functional unit that has been evolutionarily conserved (see ‘conserved elements in the 3’ UTR).
The 3'-UTR in the human and feline sequences are more than 100 nucleotides longer than the other 3' UTR sequences, with the exception of the gallus PTH mRNA.
Large gaps have to be introduced to maximize homology to the human 3'-UTR.
Hendy et al suggested that the extra sequence in the 3’ region of the human cDNA, corresponding to the large gap in the bovine sequence, might have been the result of a gene duplication since it contained some homology to the region around the polyadenylation signal, including a second consensus polyadenylation signal.
Interestingly, in the rat sequence, large gaps also must be introduced in this region, but they do not coincide exactly with that of the bovine sequence.
The same phylogenetic tree is obtained from the amino acid sequences and from the coding regions of the mRNA.
Phylogenetic comparison based on nt similarity of the full PTH mRNAs or the 3'-UTRs.
This map does not !!include!! macaca and equine PTH sequences where there are only partial sequences of the cDNA available.
The gallus is very different from all the other species indicating a separate evolutionary branch. Interestingly, based on amino acid sequence and the coding region of the mRNA, the bovine and porcine were grouped closest to canis and felis but not by the full-length mRNA or 3'-UTR sequences.
This mainly represents the largedifferences in the 3'-UTRs and correlates with the conservation of protein-binding elements. The mouse and rat species are separate because they only have the distal PTH mRNA 3'-UTR element.
The human, canis, felis, bovine and porcine are grouped together, all containing the proximal element.
But in this group, the bovine and porcine represent a separate branch expressing only the proximal element, and the human, felis and canine are a distinct branch, which corresponds with their expression of both the proximal and distal elements.
